Backscattered Electron Imaging of Colloidal Gold Markers For Leukocyte Surface Antigens

1985 ◽  
Vol 43 ◽  
pp. 534-537
Author(s):  
Etienne de Harven ◽  
Davide Soligo
Keyword(s):  
Colloidal Gold ◽  
Bacteriophage T4 ◽  
Small Cell ◽  
Surface Structures ◽  
Coated Pits ◽  
Backscattered Electron Imaging ◽  
Cell Surface Structures ◽  
Backscattered Electron ◽  
Electron Imaging ◽  
20 Nm

Markers for immuno-scanning electron microscopy had been, so far, selected for easy identification based on their distinctive shape (for example, haemocyanin, bacteriophage T4). These markers were always viewed in the secondary electron imaging (SEI) mode. Their size was not posing much of a problem of resolution for commercially available SEM, but was likely to prevent good labeling efficiency, due to steric hindrance phenomena. Higher labeling efficiency necessitates the use of markers of smaller size which, unfortunately, can rarely be unambiguously recognized in topographical SE images.To correlate antigenic distribution with small cell surface structures like microvilli or coated pits, markers of smaller size (i.e., in the 20 nm range) must be used and clearly identified on the surface of well preserved cells. With the availability of colloidal gold particles complexed with various ligands this became possible, particularly if the labeled cells are viewed in the backscattered imaging (BEI) mode of the SEM, therefore basing the identification of the marker on its atomic number contrast rather than on its topographical contrast.

Application of Backscattered Electron Imaging to the Study of Scavenger Receptor-Mediated Endocytosis by Sinusoidal Cells in Bone Marrow

1985 ◽  
Vol 43 ◽  
pp. 542-545
Author(s):  
R.P. Becker ◽  
J.S. Geoffroy
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Coated Pits ◽  
Thin Sections ◽  
Backscattered Electron Imaging ◽  
Sinusoidal Cells ◽  
Dense Bodies ◽  
Sinusoidal Cell ◽  
Backscattered Electron ◽  
Electron Imaging

The endothelial cells lining the postcapillary venous sinuses (sinusoids) in bone marrow take up colloidal gold-bovine serum albumin (BSA-Au) conjugates by means of a pathway involving coated pits and vesicles. Endocytosis of BSA- Au by these sinusoidal endothelial cells (sinusoidal cells) is rapid. Within one minute of pulse presentation (5 sec; intraaortic injection) with BSA-Au the probe is internalized and processed through pleomorphic endosomes to dense bodies known to be secondary lysosomes. By this time, 17% of the sinusoidal cell related BSA-Au is associated with the surface, while 83% is internalized, of which 2% is present in lysosomes. By four minutes, less than 8% of the observed BSA-Au is not internalized, the bulk being present predominantly in large pleomorphic vacuoles and dense bodies.That the endocytic process involves coated pits and vesicles prompts the suggestion that it may be receptor mediated. In order to investigate this possibility, biochemical and morphological studies were performed to determine the specificity and saturability of the putative receptor. Morphological analysis of TEM thin sections was aided by viewing large areas of the luminal sinusoidal cell surface in secondary electron (SEI) and backscattered electron imaging (BEI) modes of the scanning electron microscope.

Backscattered-electron imaging of the colloidal gold surface marker

1984 ◽  
Vol 42 ◽  
pp. 254-255
Author(s):  
E. de Harven ◽  
R. Leung ◽  
H. Christensen
Keyword(s):  
Colloidal Gold ◽  
High Efficiency ◽  
Human Peripheral Blood ◽  
Gold Surface ◽  
Blood Leukocytes ◽  
Backscattered Electron Imaging ◽  
Carbon Coated ◽  
Backscattered Electron ◽  
Electron Imaging ◽  
Gold Marker

A variety of markers have been used for surface labeling with the scanning electron microscope (1). They are all recognized in the secondary electron imaging (SEI) mode on the basis of their characteristic sizes and shapes. This makes it difficult, however, to recognize unambiguously markers of small size (smaller than 40 nm). Unfortunately, markers of such size are needed if one wishes to minimize steric hindrance phenomena (2) and therefore obtain high efficiency labeling. We have found that the atomic number contrast of the colloidal gold marker, expressed in the backscattered electron imaging (BEI) mode, alleviates this difficulty to a significant extent (3).Human peripheral blood leukocytes, separated by sedimentation in presence of Dextran, were attached to poly-1-lysine pretreated carbon coated grids. The cells were then fixed for 5 min with 0.25% solution of buffered glutaraldehyde, pH 7.2, extensively rinsed with 0.1% glycine in PBS, and incubated for 30 min, at room temperature with an IgM monoclonal antibody (D2) specifically recognizing, a surface antigen of mature granulocytes.

Capturing Defects in Flip-Chip CMOS Devices Using Backside EBAC Technique and SEM Microscopy

2016 ◽  
Author(s):  
Yuanjing (Jane) Li ◽  
John Aguada ◽  
Jiafang Lu ◽  
Jessica Yang ◽  
Roy Ng ◽  
...  
Keyword(s):  
Graphics Processing Units ◽  
Flip Chip ◽  
Bulk Silicon ◽  
Backscattered Electron Imaging ◽  
Visual Defects ◽  
Backscattered Electron ◽  
Electron Imaging ◽  
Graphics Processing ◽  
20 Nm ◽  
Silicon Cmos

Abstract This paper presents backside physical failure analysis methods for capturing anomalies and defects in advanced flip-chip packaged, bulk silicon CMOS devices. Sample preparation involves chemically removing all the silicon, including the diffusions, to expose the source/drain contact silicide and the gate of the transistors from the backside. Scanning Electron Microscopy (SEM) is used to form high resolution secondary and/or backscattered electron images of the transistor structures on and beneath the exposed surface. If no visual defects/anomalies are found at the transistor level, the Electron Beam Absorbed Current (EBAC) technique is used to isolate short/open defects in the interconnect metallization layers by landing nano-probe(s) on a transistor’s source/drain silicide or on the gate. Using the combination of secondary and backscattered electron imaging and backside EBAC thus allows defects residing in either the transistors or the metal nets to be found. Case studies from 20 nm technology node graphics processing units (GPU) are presented to demonstrate the effectiveness of this approach.

High-sensitivity detection of gold labels by high-angle dark-field STEM imaging

1990 ◽  
Vol 48 (3) ◽  
pp. 892-893 ◽  
Author(s):  
Max T. Otten
Keyword(s):  
High Sensitivity ◽  
Dark Field ◽  
Bright Field ◽  
Backscattered Electron Imaging ◽  
Backscattered Electrons ◽  
Average Atomic Number ◽  
Highly Sensitive ◽  
Backscattered Electron ◽  
Electron Imaging ◽  
High Sensitivity Detection

Labelling of antibodies with small gold probes is a highly sensitive technique for detecting specific molecules in biological tissue. Larger gold probes are usually well visible in TEM or STEM Bright-Field images of unstained specimens. In stained specimens, however, the contrast of the stain is frequently the same as that of the gold labels, making it virtually impossible to identify the labels, especially when smaller gold labels are used to increase the sensitivity of the immunolabelling technique. TEM or STEM Dark-Field images fare no better (Figs. 1a and 2a), again because of the absence of a clear contrast difference between gold labels and stain.Potentially much more useful is backscattered-electron imaging, since this will show differences in average atomic number which are sufficiently large between the metallic gold and the stains normally used. However, for the thin specimens and at high accelerating voltages of the STEM, the yield of backscattered electrons is very small, resulting in a very weak signal. Consequently, the backscattered-electron signal is often too noisy for detecting small labels, even for large spot sizes.

Enzyme histochemical application and backscattered electron imaging to visualize the distribution of lymphatic capillaries

1990 ◽  
Vol 48 (3) ◽  
pp. 880-881
Author(s):  
Seiji Kato
Keyword(s):  
Staining Method ◽  
Double Staining ◽  
Reaction Products ◽  
Blood Capillaries ◽  
Backscattered Electron Imaging ◽  
Histochemical Observation ◽  
Backscattered Electron ◽  
Double Staining Method ◽  
Electron Imaging ◽  
Cryostat Sections

Previously, the author repeatedly confirmed the higher 5’-nucleotidase (5’-Nase) and lower alkaline phoaphatase (ALPase) activities in the wall of lymphatic capillaries reacted with the lead-based method relative to those of blood capillaries. The ALPase, on the other hand, is markedly higher in blood capillaries than in lymphatics. On the basis of these enzyme characteristics, the author has developed a 5’-Nase— ALPase double staining method to differentiate small lymphatics from blood capillaries at the level of the light microcsopy. Furthermore, we applied it to histochemical observation of the lead-containing reaction products of 5’-Nase in lymphatics on the same or adjacent cryostat sections using backscattered electron imaging (BEI) in scanning electron microscope (SEM). This paper presents a new applicability of 5’-Nase histochemistry by BEI-SEM to demonstrate the distribution of lymphatic capillaries in tissue blocks.

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